The secretory biology of human neutrophils (PMN) is profoundly affected by their adhesion to biological surfaces. The aim of this grant is to better understand how CD11/CD18 integrins and physiologic peptide agonists act synergistically to trigger PMN to secrete large amounts of oxidants, and how this potentially destructive process is regulated. We focus on signalling through each of the two known types of TNF receptors (TNF-R), compared to agonists of other classes (C5a, FMLP, PMA and bacteria). For TNF (or TNF-mimetic anti-TNF-R mAbs) to induce a full- scale respiratory burst in PMN, the cells must use CD11/CD18 integrins and must interact with matrix proteins in the solid phase. A synergistic interaction between TNF-R and adhesion receptors leads to a sustained fall in cAMP. This in turn permits cell spreading and reorganization of actin into focal adhesions, changes that are essential for the full- scale respiratory burst. Other key transduction events that depends both on TNF and on adhesion are tyrosine protein phosphorylation, and activation of protein kinase C (PKC). Both processes precede the burst; inhibitors of both block the burst. The TNF-induced burst is also blocked by inhibitors of protein kinase A (PKA) and phospholipase C. Finally, PMN adhesion exerts a profound effect on the number of cell surface TNF receptors (TNF-R). PMN stimulated in suspension cleave p75 TNF-R with elastase, and cleave both p55 and p75 TNF-R with an apparently novel protease. In contrast, adherent PMN maintain their cell surface TNF-Rs by mobilizing elastase-resistant p55 TNF-R from specific granules, and by inhibiting the novel receptorase. Thus, we have identified 3 newly recognized determinants of the adhesion-dependent respiratory burst of PMN: activation of three protein kinase systems (PKY, PKC, PKA); control of TNF-R function by cross-linking and by reorganization of the actin cytoskeleton; and proteolysis of TNF-Rs by suspended but not adherent cells, such that only suspended PMN lose responsiveness to TNF and generate soluble TNF inhibitors. The proposed experiments address: (1) The transducing role of protein kinases. The main goal is identification of protein tyrosine kinase (PKY) hypothesized to associate with TNF-R. (2) The intermediary role of cytoskeletal reorganization. We will confirm the critical role of TNF-R crosslinking, and assess the cytoskeletal association and molecular co- localization of TNF receptors, CD11/CD18 integrins, and oxidase components in PMN spreading on protein-coated surfaces. (3) The regulatory role of receptor-shedding proteases. We aim to purify and clone the novel receptorase that cleaves p55 and p75 TNF-R's, and analyze how adhesion regulates its action. This may shed light on the proteases that regulate cell surface receptors on many types of cells. The proposed experiments provide a rare opportunity to study signal transduction separately and together through each of the two types of TNF receptors in the same cell population, and more broadly, to study how a nontransformed population of human cells mounts a rapid, functionally important response to a cytokine without gene transcription or protein synthesis, in a manner controlled jointly by two distinct sets of receptors (TNF-Rs and integrins).